1.1 Field of the Invention
The present invention relates to gene expression in mammalian cells. More particularly, the invention relates to a genetic element which provides for cytoplasmic expression of intron containing cellular mRNA which normally do not exit the nucleus. Nucleic acid constructs containing a gene normally transcribed into intron containing mRNA is located proximal to this genetic element such that the result of transcription from this gene is an intron containing mRNA transcript also containing the genetic element. The genetic element also can be used for complex retroviral protein expression and viral replication independent of the expression of a viral transacting protein such as rev, rex, or functionally equivalent protein.
1.2 Description of the Background and Related Art
A. Splicing of HIV RNA:
The human immunodeficiency virus (HIV) is a lymphotropic retrovirus implicated in the pathogenesis of AIDS. As compared to other characterized retroviruses, the HIV genome appears to contain at least six novel genes (vif, vpr, tat, rev, vpu, and nef). However, a common feature of all replication-competent retroviruses is that the primary transcription product from the proviral DNA contains at least three open reading frames gag, pol and env, positioned 5' to 3' in the RNA. This product is always a genome length RNA that is spliced to generate subgenomic species, wherein the spliced RNA function as mRNA for env or other proteins that are sometimes encoded near the 3' end of the genome. Splicing, the removal of intervening sequences, is a multi-step process requiring the participation of small nuclear RNAs and protein factors that together make up small nuclear ribonucleoprotein particles (snRNP) which in turn form a large complex termed the spliceosome.
In the case of the "simple" Oncornavirus subfamily of retroviruses, a single 5' splice site is positioned near the 5' end of the primary transcript and splicing involves the use of one or two 3' acceptor sites positioned downstream in the RNA. Thus, the subgenomic molecules are always singly spliced and have had most or all of the coding region for gag and pol removed. In these RNAs, the gag and pol region has been defined as an intron. However, because splicing is inefficient, enough full length RNA remains to function as both the mRNA for the gag and pol genes and as the molecule that is packaged into virus particles (Coffin, 1991, in Fundamental Virology, eds. Field et al., pp.645-708, Raven Press Ltd.).
The situation in HIV, a member of the Lentivirus subfamily of retroviruses, is more complex. In this case, the coding regions of several novel genes are positioned near the center of the primary transcript between gag-pol and env and at the 3' end of the genome (Wong-Staal, 1991, in Fundamental Virology, eds. Field et al., pp.709-723, Raven Press Ltd. ). The central region of the genome also contains several 5' and 3' splice sites, which, in conjunction with the conventionally positioned 5' splice site near the 5' end of the RNA, are used for differential splicing of the primary transcript into over twenty different species of mRNA (Schwartz et al., 1990, J. Virol. 64:2519-2529; Schwartz et al., 1990, J. Virol. 64:5448-5456; Schwartz et al., 1991, Virology 183:677-686). These RNAs are either singly or multiply spliced. A consequence of this complicated splicing scheme is that env, as well as gag-pol, has been defined as an intron in the multiply spliced mRNAs.
B. The Relationship Between Rev, Splicing and HIV Gene
Expression and Replication:
In most cases, cellular mRNAs contain introns that are removed by splicing before transport to the cytoplasm occurs. Transport to the cytoplasm is required for the mRNA to interact with the ribosomes and accessory factors in the process of protein synthesis. Recent studies have suggested that intron-containing RNAs are usually prevented from exiting the nucleus due to the binding of splicing factors (Chang and Sharp, 1989, Cell 59:789-795; Legrain and Rosbash, 1989, Cell 57:573-583); although there are a few examples of differentially spliced cellular transcripts that are transported with a retained intron (McKeown, 1992, Annual Rev. of Cell Biol. 8:133-155). While the exact export mechanism(s) that allow these mRNAs to be transported has yet to be elucidated, recent data indicates that different RNA species may share common steps in the export pathway. In that regard, export is believed to involve targeting to the sites of exit and transport through them, probably as a RNA-protein complex. Thus, the cellular pathway of nucleocytoplasmic transport of mRNA includes a factor (one or more proteins), intrinsic to the cell, which binds to a sequence(s) contained in the mRNA.
The rev gene has been shown to be essential for the production of virus (herein "rev" refers to the gene and "rev" refers to the gene product; this convention is also followed for other gene/protein pairs such as env/env, etc.). Using infectious proviral clones of HIV to study rev function, it has been demonstrated that mutations in this gene led to severely reduced levels of protein from gag and env (Feinberg et al., 1986, Cell 46:807-817; Sodroski et al., 1986 Nature 321:412-417). In these studies, in the absence of rev, the levels of large mRNAs encoding the structural proteins were reduced, whereas the levels of doubly spliced small RNAs encoding nonstructural proteins were increased. Similarly, using an envelope protein expression vector system, when rev was deleted from the vector, steady-state levels of env mRNA in the cytoplasm were greatly reduced; env RNA accumulated in the nucleus; and no env protein could be detected unless rev was provided in trans (Hammarskjold et al., 1989, J. Virol. 63:1959-1966).
It has been shown that the HIV rev protein functions to specifically allow nuclear export of unspliced and singly spliced HIV RNA (Emerman et al., 1989, Cell 57:1155-1165; Felber et al., 1989, Proc. Natl. Acad. Sci. USA 86:1496-1499); Hammarskjold et al., 1989, supra; Malim et al., 1989, Nature 338:254-257). These RNAs contain complete introns and are retained in the nucleus in the absence of rev. The details of how rev functions are not completely elucidated, although it is clear that rev action requires it to bind to a specific element in the HIV RNA known as the rev responsive element (RRE) (Daly et al., 1989 Nature 342:816-819; Hammarskjold et al., 1989, supra; Zapp and Green, 1989, Nature 342:714-716). It is believed that rev then acts to allow export of unspliced RNA by interacting with a cellular protein termed a "rev-interacting protein" (Gorlich et al., 1996, Science 271:1513-1518).
Another subfamily of complex Retroviruses, typified by HTLV I and II, seems to have evolved a mechanism similar to HIV to facilitate the transport of intron-containing RNA. These viruses utilize a protein called rex, which, like rev, must bind to a specific element present in the viral RNA (RXRE) (Ahmed et al., 1990, Genes Dev. 4:1014-1022). Rex has also been shown to substitute for rev in promoting the transport of rev-dependent mRNA (Rimsky et al., 1988, Nature 335:738-740; Lewis et al., 1990, J. Virol. 64:1690-1697). While the complex retroviruses have developed rev and rex regulation to allow the cytoplasmic expression of their intron-containing RNA, the simple retroviruses appear not to have similar transacting proteins. Rather, as shown by the present invention, simple retroviruses tap into a constitutive cellular pathway by interacting with a cellular factor or factors that may normally be used for the transport of cellular mRNA from the nucleus to the cytoplasm.
C. Relevance to Therapy against AIDS:
An important aspect in identifying anti-viral compounds that are effective against HIV is the development of in vitro assays that can be used to screen for agents that selectively interfere with the different processes involved in HIV infection and replication. One such assay, described in U.S. Pat. No. 4,910,132 involves a virus-free assay that tests the ability of compounds to inhibit specifically the synthesis of the HIV gp120 envelope protein. Another assay using recombinant vectors was developed to detect agents that would inhibit fusion between env producing cells and CD4.sup.+ cells (Nelson et al., 1989, Vth International AIDS Conference, Montreal, Quebec, Canada). However, these assays employed env-producing vectors that also encoded rev, because, inter alia, rev is required for env synthesis. Thus, in vitro assays such as these aren't able to distinguish between compounds acting to inhibit processes involving gp 120 from compounds affecting rev activity. Similarly, in vitro assays, using subgenomic constructs for the production of gag or gag-pol, are not able to distinguish between compounds acting to inhibit processes involving these proteins from compounds affecting rev activity.
Much research has been focused on the development of a vaccine against AIDS, particularly a vaccine that can readily elicit significant levels of neutralizing antibodies that would prevent the debilitating effects of HIV infection. Vaccine candidates include inactivated virus (see for example, Gibbs et al., 1991, Proc. Natl. Acad. Sci. USA 88:3348-52), virus-like particles (Smith et al., 1990, J. Virol. 64:2743-2750), gag/env protein (U.S. Pat. No. 4,925,784), recombinant fusion polypeptides containing HIV envelope protein or portions thereof (U.S. Pat. No. 5,130, 248), glycosylated envelope protein (U.S. Pat. No. 4,725,669), and envelope peptides (U.S. Pat. No. 4,957,737). However, for human use, there is yet to be demonstrated a safe and effective vaccine against HIV (Sabin, 1992, Proc. Natl. Acad. Sci. USA 89:8852-8855; Hilleman, 1992, AIDS Res. Hum. Retroviruses 8:1743-1747; Ada et al. 1992, Nature 359:572; and Desrosiers, 1992, AIDS Res. Hum. Retroviruses 8:411-421). A new approach, proposing the development of a simpler retroviral vaccine against HIV, is based on the general observation that mammalian immune systems are much more successful in controlling infection caused by simpler retroviruses, as opposed to infections by more complex retroviruses such as HIV (Temin, 1993, Proc. Natl. Acad. Sci. USA 90:4419-4420). Thus, the development of a simplified HIV may result in a virus limited in replication such that an infected human may be able to respond by successfully mounting a protective response which would also be effective against wild type HIV. "Simplified" means that this engineered virus would express only the gag, pol, and env proteins. However, an obstacle to the development of such a HIV vaccine is that env production and viral replication is dependent on the presence of rev.